Method of sequencing dna

ABSTRACT

The present invention provides a method of identifying a base at a target position in a sample nucleic acid sequence, said method comprising: subjecting a primer hybridised to said sample nucleic acid immediately adjacent to the target position, to a polymerase primer extension reaction in the presence of a nucleotide, whereby the nucleotide will only become incorporated if it is complementary to the base in the target position, and determining whether or not said nucleotide is incorporated by detecting whether Ppi is released, the identity of the target base being determined from the identity of any nucleotide incorporated, wherein, where said nucleotide comprises an adenine base, an α-thio triphosphate analogue of said nucleotide is used, ant the Rp isomer of said analogue and/or the degradation products of said analogue are eliminated from the polymerase reaction step.

[0001] This invention relates to improvements in methods of sequencingDNA, based on the detection of base incorporation by the release ofpyrophosphate (PPi).

[0002] DNA sequencing is an essential tool in molecular geneticanalysis. The ability to determine DNA nucleotide sequences has becomeincreasingly important as efforts have proceeded to determine thesequences of the large genomes of humans and other organisms.

[0003] Techniques enabling the rapid detection of a single DNA basechange, or a few base changes, are also important tools for geneticanalysis, for example in clinical situations in the analysis of geneticdiseases or certain cancers. Indeed, as more and more diseases arediscovered to be associated with changes at the genetic level, mostnotably single nucleotide polymorphisms (SNPs), the need for methods ofboth screening for SNPs or other mutations or genetic changes (bysequencing representative genomic samples) and scoring SNPs (or othermutations/changes) grows. Thus, as well as the development of novelsequencing technologies for determining the sequence of longer stretchesof DNA, the art has also seen a rapid rise in the development oftechnologies for detecting single (or a few) base changes. Suchprotocols to determine more limited sequence information, relating toonly one or a few bases are termed mini-sequencing.

[0004] The method most commonly used as the basis for DNA sequencing, orfor identifying a target DNA base, is the enzymatic chain-terminationmethod of Sanger. Traditionally, such methods relied on gelelectrophoresis to resolve, according to their size, DNA fragmentsproduced from a larger DNA segment. However, in recent years varioussequencing technologies have evolved which rely on a range of differentdetection strategies, such as mass spectrometry and array technologies.

[0005] One class of sequencing methods assuming importance in the artare those which rely upon the detection of PPi release as the detectionstrategy. It has been found that such methods lend themselves admirablyto large scale genomic projects or clinical sequencing or screening,where relatively cost-effective units with high throughput are needed.

[0006] Methods of sequencing based on the concept of detecting inorganicpyrophosphate (PPi) which is released during a polymerase reaction havebeen described in the literature for example (WO 93/23564, WO 89/09283,WO98/13523 and WO 98/28440). As each nucleotide is added to a growingnucleic acid strand during a polymerase reaction, a pyrophosphatemolecule is released. It has been found that pyrophosphate releasedunder these conditions can readily be detected, for example enzymicallye.g. by the generation of light in the luciferase-luciferin reaction.Such methods enable a base to be identified in a target position and DNAto be sequenced simply and rapidly whilst avoiding the need forelectrophoresis and the use of labels.

[0007] At its most basic, a PPi-based sequencing reaction involvessimply carrying out a primer-directed polymerase extension reaction, anddetecting whether or not that nucleotide has been incorporated bydetecting whether or not PPi has been released. Conveniently, thisdetection of PPi-release may be achieved enzymatically, and mostconveniently by means of a luciferase-based light detection reactiontermed ELIDA (see further below).

[0008] It has been found that dATP added as a nucleotide forincorporation, interferes with the luciferase reaction used for PPidetection. Accordingly, a major improvement to the basic PPi-basedsequencing method has been to use, in place of dATP, a dATP analogue(specifically dATPαs) which is incapable of acting as a substrate forluciferase, but which is nonetheless capable of being incorporated intoa nucleotide chain by a polymerase enzyme (WO98/13523).

[0009] Further improvements to the basic PPi-based sequencing techniqueinclude the use of a nucleotide degrading enzyme such as apyrase duringthe polymerase step, so that unincorporated nucleotides are degraded, asdescribed in WO 98/28440, and the use of a single-stranded nucleic acidbinding protein in the reaction mixture after annealing of the primersto the template, which has been found to have a beneficial effect inreducing the number of false signals, as described in WO00/43540.

[0010] However, even with the modified and improved PPi-based sequencingmethods mentioned above, there is still room for improvement, forexample to increase the efficiency and/or accuracy of the procedure, or,as discussed further below, to increase the sequence read lengthpossible. The present invention addresses these needs.

[0011] In particular, the present invention is concerned with methods ofPPi-based sequencing which use an α-thio analogue of deoxy ATP (dATP)(or dideoxy ATP (ddATP)) namely an (1-thio) triphosphate (orα-thiophosphate) analogue of deoxy or dideoxy ATP, preferablydeoxyadenosine [1-thio] triphosphate or deoxyadenosineα-thiotriphosphate (dATPαs) as it is also known. dATPαs (as with allα-thio nucleotide analogues) occurs as a mixture of isomers, the Rpisomer and the Sp isomer.

[0012] When dATPαS (and/or other α-thio nucleotides) are used, it hasbeen found that the efficiency of the sequencing method decreases as thenumber of cycles increases, and in particular that the read lengthattainable is limited (e.g. to 40-50 bases). This is believed to be dueto the accumulation of inhibitory substances in the reaction system. Thepresent invention is particularly concerned with reducing or removingsuch inhibitory effects.

[0013] More particularly, it has surprisingly been found that removingor excluding the Rp isomer of α-thio nucleotide analogues and/or thedegradation products of said analogue from the polymerisation mixtureimproves the efficiency of PPi-based sequencing methods, and inparticular, such methods based on detecting PPi by a luciferase-basedELIDA reaction.

[0014] Whilst not wishing to be bound by theory, it is believed that anumber of reasons or effects may contribute to this beneficial effect ofRp isomer and/or the degradation products of the α-thio nucleotideanalogue elimination on the sequencing reaction. Certain of thoseeffects are explained further below, but in particular we believe thatthe Rp isomer and/or the degradation products of the α-thio nucleotideanalogue are capable of inhibiting polymerase activity. Thus, it isbelieved that one such contributory effect may be due to the effect ofthe Rp isomer and/or the degradation products of the α-thio nucleotideanalogue, not hitherto appreciated, in inhibiting the activity ofpolymerase. Hence, by excluding or removing the Rp isomer and/or thedegradation products of the α-thio nucleotide analogue, this inhibitoryeffect may be avoided, leading to a more efficient and fasterpolymerisation reaction, and more even signals generated in thePPi-detection reaction. Further, by removing or excluding the Rp isomerand/or the degradation products of the α-thio nucleotide analogue, thefidelty of the DNA synthesis is increased, i.e. the number ofmisincorporation events is decreased.

[0015] In one aspect, the present invention thus provides a method ofidentifying a base at a target position in a sample nucleic acidsequence, said method comprising:

[0016] subjecting a primer hybridised to said sample nucleic acidimmediately adjacent to the target position, to a polymerase primerextension reaction in the presence of a nucleotide, whereby thenucleotide will only become incorporated if it is complementary to thebase in the target position, and determining whether or not saidnucleotide is incorporated by detecting whether PPi is released, theidentity of the target base being determined from the identity of anynucleotide incorporated,

[0017] wherein, where said nucleotide comprises an adenine base, anα-thio triphosphate analogue of said nucleotide is used, and the Rpisomer of said analogue and/or the degradation products of said analogueare eliminated from the polymerase reaction step.

[0018] Use of α-trio triphosphate analogue of a nucleotide comprising anadenine base and eliminating the Rp isomer thereof and/or eliminatingthe degradation products of said analogue are essential features of thepresent invention, which this does not relate to, e.g. minisequencingreactions where only nucleotides comprising guanine, thymine or cytosinebases are added for incorporation.

[0019] Subject to the proviso that where the nucleotide comprises anadenine base (A), an α-thio triphosphate analogue of said nucleotide isused, the nucleotide may be any nucleotide capable of incorporation by apolymerase enzyme into a nucleic acid chain or molecule. Thus, forexample, the nucleotide may be a deoxynucleotide (dNTP, deoxynucleosidetriphosphate) or dideoxynucleotide (ddNTP, dideoxynucleosidetriphosphate).

[0020] Conveniently, for sequencing purposes, guanine (G), cytosine (C),thymine (T) or adenine (A) deoxy- or dideoxy-nucleotides may be used.Thus the nucleotide may be dGTP (deoxyguanosine triphosphate), dCTP(deoxycytidine triphosphate) or dTTP (deoxythymidine triphosphate) or inplace of dATP (deoxyadenosine triphosphate) its α-thiotriphosphateanalogue, dATPαs (deoxyadenosine α-thiotriphosphate ordeoxyadenosine[1-thio]-triphosphate as it is also known). Analogously,the nucleotides may be ddGTP, ddCTP, or dTTP, or in place of ddATP,ddATPαs.

[0021] The present invention thus requires the use of anα-thiotriphosphate analogue of an adenine nucleotide, but for the otherbases (G, T or C), a native nucleotide may be used (or indeed any othernucleotide, e.g. nucleotide derivative, it is desired to use, providedthat it can be incorporated by a polymerase enzyme). Thus, according tothe present invention, at least an α-thio analogue of an adeninenucleotide is used.

[0022] However, it may in certain cases be desirable also to use α-thioanalogues of one or more other nucleotides (i.e. of guanine, cytosine orthymine nucleotides). In any such case where an α-thio analogue of anucleotide is used, then according to the present invention the Rpisomer of said analogue is eliminated from the polymerase reaction step.In other words, in the method of the invention, where the nucleotide isan α-thio nucleotide (e.g. deoxy- or dideoxy-nucleosideα-thiotriphosphate (dNTPαS or ddNTPαS)), the Rp isomer of said α-thionucleotide is eliminated from the polymerase reaction step, and /or thedegradation products of the NTPαS are eliminated.

[0023] For convenience, the term “deoxynucleoside α-thiotriphosphate”(dNTPαS) as used herein thus includes deoxyadenosine α-thiotriphosphate(dATPαS), deoxycytidine α-thiotriphosphate (dCTPαS), deoxyguanosineα-thiotriphosphate (dGTPα) and deoxythymidine α-thiotriphosphate(dTTPαS). Analogously, the term “dideoxy nucleotide α-thio triphosphate”includes the dideoxy equivalent. The term “dideoxynucleotide” as usedherein includes all 2′-deoxynucleotides in which the 3′-hydroxyl-groupis absent or modified and thus, whilst being able to be added to theprimer in the presence of the polymerase, is unable to enter into asubsequent polymerisation reaction, i.e. a dideoxy nucleotide is thus a“chain terminator”, and as is well known in the art, certain sequencingmethods may employ such chain-terminating nucleotides.

[0024] The term “NTPαS” (nucleoside α-thiotriphosphate) is used hereinto refer to all α-thiotriphosphate nucleotide analogues (i.e.α-thionucleotides) which may be used according to the present invention,and includes both ribo- and deoxyribo- (or dideoxy-) nucleotides. Theseinclude, most notably, dNTPαS and ddNTPαS.

[0025] When synthesized, α-thiotriphosphate nucleotide analogues (e.g.dd- or dNTPαS) are produced typically in two isomeric forms, the Sp andRp isomers. An α-thiotriphosphate nucleotide analogue possesses a chiralcentre, and thus the 2 species are enantiomers. The Sp isomer is theleft-handed isomer, also designated the L isomer. The right-handedisomer is the Rp isomer, also known as the D-isomer (see e.g. Eckstein(1985), Ann. Rev. Biochem., 54: (367-402).

[0026] The Rp isomer of NTPαS does not act as a substrate for apolymerase enzyme, as used in the primer extension reaction.Surprisingly, however, as mentioned above, it has been found that use ofthe Rp isomer of NTPαS leads to inhibition of enzymes involved in thePPi-based sequencing reactions. The precise nature of these variousinhibitory effects is not entirely clear, but it is believed that the Rpisomer of NTPαS itself (and possibly also the Rp isomer of NDPαS and Rpisomer of NMPαS) are responsible for the inhibitory effects observed,including, as mentioned above, inhibition of polymerase. The degradationproducts of NTPαS are also responsible for the inhibitory effects seen.The degradation products include, but are not limited to, NDPαS, NMPαSand any degradation products thereof. The inhibitory effects of the Rpisomer of dATPαS on nucleotide degrading enzymes and PPi detectionenzymes have been investigated (see Example 2) and FIGS. 8I, 8J, 8K and8L) and it has been shown that the Rp isomer and/or degradation productsof the dATPαS do inhibit enzymes involved in nucleotide degaradation anddetection of PPi release.

[0027] It will be appreciated that when the target base immediately 3′-of the primer has an identical base 3′-thereto, and the polymerisationis effected with a deoxynucleotide (rather than a dideoxynucleotide) theextension reaction will add two bases at the same time and indeed anysequence of successive identical bases in the sample will lead tosimultaneous incorporation of corresponding bases into the primer.However, the amount of pyrophosphate liberated will clearly beproportional to the number of incorporated bases so that there is nodifficulty in detecting such repetitions.

[0028] Since the primer is extended by a single base by the proceduredescribed above (or by a sequence of identical bases), the extendedprimer can serve in exactly the same way in a repeated procedure todetermine the next base in the sequence, thus permitting the wholesample to be sequenced.

[0029] The method of the invention may thus be used to determine theidentity (i.e. sequence) of a single base. However, conveniently, byrepeating the primer extension steps in the presence of a further(successive) nucleotide, the sequence (or identity) of a further base inthe sequence of the sample nucleic acid may be revealed. Accordingly,the method of the invention may be used to determine the identity of oneor more bases in a sample nucleic acid (i.e. to determine the sequenceof one or more bases in a sample nucleic acid).

[0030] The method of the invention thus has utility in a number ofdifferent sequencing methods and formats, including mini-sequencingprocedures e.g. detection of single base changes (for example, indetecting point mutations, or polymorphisms, or allelic variations etc).Accordingly, the method of the invention may thus be used in a “full”sequencing procedure, i.e. the identification of the sequential order ofthe bases in a stretch of nucleotides, as well in single base detectionprocedures.

[0031] For example, to determine sequence information in a targetnucleotide sequence (i.e. target or sample nucleic acid), differentnucleotides may be added either to separate aliquots of sample-primermixture (e.g. four aliquots, one for each of the four, A, T, G or Cnucleotides) or successively to the same sample-primer mixture andsubjected to the polymerase reaction to indicate which nucleotide isincorporated.

[0032] In order to sequence the sample nucleic acid, the procedure maybe repeated one or more times i.e. cyclically, as is known in the art.In this way the identity of several or many bases in the sample nucleicacid may be identified, essentially in the same reaction.

[0033] Where separate aliquots are used, once it has been identifiedwhat base has been incorporated (i.e. in which aliquot incorporation hastaken place), the “incorporated” base may be added to the “unreacted”aliquots, to extend the primer in all aliquots, before repeating theprocess (cycling) to sequence the next base. In the “successive”embodiment, a different nucleotide may be added successively untilincorporation is indicated by PPi release, whereupon the procedure maybe repeated.

[0034] Hence, a sequencing protocol may involve annealing a primer asdescribed above, adding a nucleotide, performing a polymerase-catalysedprimer extension reaction, detecting the presence or absence ofincorporation of said nucleotide by detecting any PPi released, andrepeating the nucleotide addition and primer extension steps etc. one ormore times. As discussed above, single (i.e. individual) nucleotides maybe added successively to the same primer-template mixture, or toseparate aliquots of primer-template mixture, etc. according to choice,and the sequence information it is desired to obtain.

[0035] In order to permit the repeated or successive (iterative)addition of nucleotides in a multiple-base sequencing procedure, thepreviously-added nucleotide must be removed. This may be achieved bywashing, or more conveniently, by using a nucleotide-degrading enzyme,for example as described in detail in WO98/28440.

[0036] Accordingly, in a principal embodiment of the present invention,a nucleotide degrading enzyme is used to degrade any unincorporated orexcess nucleotide. Thus, if a nucleotide is added which is notincorporated (because it is not complementary to the target base), orany added nucleotide remains after an incorporation event (i.e. excessnucleotides) then such unincorporated nucleotides may readily be removedby using a nucleotide-degrading enzyme. This is described in detail inWO98/28440.

[0037] As will be discussed in more detail below, it has been observedthat inhibitory effects due to the use of an α-thio nucleotideparticularly occur (or are observed) when a nucleotide degrading enzymeis used, and that such effects may be beneficially abrogated accordingto the present invention, by the methods described herein.

[0038] The term “nucleotide degrading enzyme” as used herein includesany enzyme capable of specifically or non-specifically degradingnucleotides, including at least nucleoside triphosphates (NTPs), butoptionally also di- and mono-phosphates, and any mixture or combinationof such enzymes, provided that a nucleoside triphosphatase or otherNTP-degrading activity is present. Although nucleotide-degrading enzymeshaving a phosphatase activity may conveniently be used according to theinvention, any enzyme having any nucleotide or nucleoside degradingactivity may be used, e.g. enzymes which cleave nucleotides at positionsother than at the phosphate group, for example at the base or sugarresidues. Thus, a nucleoside triphosphate degrading enzyme is essentialfor the invention. Nucleoside di- and/or mono-phosphate degradingenzymes are optional and may be used in combination with a nucleosidetri-phosphate degrading enzyme. A phosphatase used as a nucleotidedegrading enzyme according to this aspect of the invention should meetseveral criteria, notably the inhibitory constant (Ki) for the phosphate(Pi) product of phosphatase action, should not be too low, so that theenzyme is not inhibited by accumulating phosphate. Secondly, the enzymeneeds to act relatively fast and should not be too slow (certainphosphatase enzymes can act too slowly to be practical) and thirdly itshould degrade all four nucleotide substrates (i.e. A, T, G and Csubstrates) with more or less equal efficiency. As discussed furtherbelow, ATP generated in the ELIDA reactions is preferred for PPidetection, and a nucleotide degrading enzyme useful in the inventionshould also efficiently degrade ATP. This leads to an efficient“turning-off” of the signal. It will be noted that not all phosphataseenzymes (e.g. alkaline phosphatases that are strongly inhibited by theproduct phosphate) meet these criteria, and so not all may be suitablefor use as a nucleotide degrading enzyme according to the invention.However, such suitability may readily be assessed by routineexperiments. The preferred nucleotide degrading enzyme is apyrase, whichis both a nucleoside diphosphatase and triphosphatase, catalysing thereactions NTP→NDP+Pi and NDP→NMP+Pi (where NTP is a nucleosidetriphosphate, NDP is a nucleoside diphosphate, NMP is a nucleotidemonophosphate and Pi is inorganic phosphate). Apyrase may be obtainedfrom the Sigma Chemical Company. Other possible nucleotide degradingenzymes include Pig Pancreas nucleoside triphosphate diphosphorydrolase(Le Bel et al., 1980, J. Biol. Chem., 255, 1227-1233). Further enzymesare described in the literature.

[0039] The nucleotide-degrading enzyme may conveniently be includedduring the polymerase (i.e. primer extension) reaction step. Thus, forexample the polymerase reaction may conveniently be performed in thepresence of a nucleotide-degrading enzyme. Although less preferred suchan enzyme may also be added after nucleotide incorporation (ornon-incorporation) has taken place, i.e. after the polymerase reactionstep.

[0040] Thus, the nucleotide-degrading enzyme (e.g. apyrase) may be addedto the polymerase reaction mixture (i.e. sample nucleic acid, primer andpolymerase) in any convenient way, for example prior to orsimultaneously with initiation of the reaction, or after the polymerasereaction has taken place, e.g. prior to adding nucleotides to thesample/primer/polymerase to initiate the reaction, or after thepolymerase and nucleotide are added to the sample/primer mixture.

[0041] Conveniently, the nucleotide-degrading enzyme may simply beincluded in the reaction mixture for the polymerase reaction, which maybe initiated by the addition of the nucleotide.

[0042] A further surprising feature of the present invention is theobservation that use of the Rp isomer of α-thio nucleotides and/ordegradation products of said α-thio nucleotides lead to inhibition of anucleotide-degrading enzyme of the present invention, most notablyapyrase. It is believed that the Rp isomer is not capable of acting as asubstrate for apyrase, and accordingly that it is not degraded by thenucleotide-degrading activity of the apyrase. Alternatively, products ofNTPαS degradation may be inhibitory. Thus, a situation may be created ofsuccessive accumulation of the inhibitory and inactive Rp isomer (or ofinhibitory degradation products) during the sequencing procedure. Thus,a further benefit derivable from the elimination of the Rp isomer and/ordegradation products of said α-thio nucleotides according to the presentinvention, is that inhibition of the apyrase may be reduced or avoided.This has very significant, and heretofore unpredictable, benefits on theefficiency and performance of the sequencing method, and represents asignificant and surprising advantage of the present invention.

[0043] Degradation of the unincorporated nucleotides by apyrase has thebenefit of producing even and well defined (e.g. narrow/defined orsharp) PPi detection signals (see further below). Where such degradationis inefficient, due to inhibition of the apyrase enzyme, this benefit isprogressively lost. Thus, inhibition of apyrase is seen, directly orindirectly, as a slower nucleotide degradation rate, and consequently asa “wider” or less well-defined PPi-detection signal, in the later cyclesof sequencing. In particular, in the context of ELIDA detection of PPirelease, described further below, the decay in signal observed (light)is “seen” as degradation of ATP (generated in the ELIDA reactions).Thus, a slower rate of degradation of ATP is “seen”. It can be inferredfrom this that the rate of unincorporated nucleotide degradation is alsoslower. Further, non-synchronised extension may result. This occurs,since the presence of undegraded unincorporated nucleotides may lead tomultiple extension reactions occurring out of phase, and leading tooverlapping signals (signal overlay or out of phase signals). Suchnon-synchronised extension thus limits the number of nucleotides whichmay be sequenced in a given sequencing run i.e. the read-lengthattainable.

[0044] The ability to sequence longer stretches of nucleic acid is adesirable goal in the sequencing field. The inhibitory effects resultingfrom the use of α-thio nucleotides on the enzymes present within thePPi-based sequencing methods of the present invention, leads todecreased fidelity and non-synchronised extension, thus limiting theread length attainable, i.e. the length of the nucleic acid which can besuccessfully sequenced. Read-length may be improved according to thepresent invention by eliminating the Rp isomer of the α-thionucleotideand/or degradation product of α-thio nucleotides.

[0045] Attainable read-length is to some extent a parameter dependentupon the acceptance criteria adopted. Cleanness of signal may be lost,but the sequence may nonetheless still be readable to a skilled andexperienced practitioner. Precise read-length limits therefore are notalways meaningful or cannot be applied generally, and may depend oncircumstances. However, it has in certain cases been found that 50 ormore, or even 100 or more (e.g. 200 or more) bases may readily be readaccording to the present invention. The methods of the invention arethus suitable for the sequencing of 100 bases or more. In particular,the present invention may advantageously be used in the sequencing of 50or more, 60 or more, 70 or more or 80 or more bases.

[0046] PPi release may be detected according to the present invention inany desired or convenient way. PPi can be determined by many differentmethods and a number of enzymatic methods have been described in theliterature (Reeves et al., (1969), Anal. Biochem., 28, 282-287; Guilloryet al., (1971), Anal. Biochem., 39, 170-180; Johnson et al., (1968),Anal. Biochem., 15, 273; Cook et al., (1978), Anal. Biochem. 91,557-565; and Drake et al., (1979), Anal. Biochem. 94, 117-120).

[0047] It is preferred to use a luciferase-based (e.g. a luciferinluciferase-based) light generating reaction to detect the release ofpyrophosphate since the amount of light generated is substantiallyproportional to the amount of pyrophosphate released which, in turn, isdirectly proportional to the number of bases incorporated. The amount oflight can readily be estimated by a suitable light sensitive device suchas a luminometer.

[0048] Luciferase-based reactions to detect the release of PPi are wellknown in the art. In particular, a method for detecting PPi releasebased on the enzymes ATP sulphurylase and luciferase has been developedby Nyrén and Lundin (Anal. Biochem., 151, 504-509, 1985) and termedELIDA (Enzymatic Luminometric Inorganic Pyrophosphate Detection Assay).The use of the ELIDA method to detect PPi is preferred according to thepresent invention. The method may however be modified, for example bythe use of a more thermostable luciferase (Kaliyama et al., 1994,Biosci. Biotech. Biochem., 58, 1170-1171) and/or ATP sulfurylase (Ondaet al., 1996, Bioscience, Biotechnology and Biochemistry, 60:10,1740-42). This method is based on the following reactions:

[0049] Accordingly, it is preferred to detect PPi release enzymatically,and the preferred detection enzymes used in the PPi detection reactionare ATP sulphurylase and luciferase.

[0050] In a PPi detection reaction based on the enzymes ATP sulphurylaseand luciferase, the signal (corresponding to PPi released) is seen aslight. The generation of the light can be observed as a curve known as apyrogram. Light is generated by luciferase action on the product, ATP(produced by a reaction between PPi and APS (see below) mediated by ATPsulphurylase) and, where a nucleotide-degrading enzyme such as apyraseis used, this light generation is then “turned off” by the action of thenucleotide-degrading enzyme, degrading the ATP which is the substratefor luciferase. The slope of the ascending curve may be seen asindicative of the activities of DNA polymerase (PPi release) and ATPsulphurylase (generating ATP from the PPi, thereby providing a substratefor luciferase). The height of the signal is dependent on the activityof luciferase, and the slope of the descending curve is, as explainedabove, indicative of the activity of the nucleotide-degrading enzyme.

[0051] Advantageously, by including the PPi detection enzyme(s) (i.e.the enzyme or enzymes necessary to achieve PPi detection according tothe enzymatic detection system selected, which in the case of ELIDA,will be ATP sulphurylase and luciferase) in the polymerase reactionstep, the method of the invention may readily be adapted to permit thesequencing reactions to be continuously monitored in real-time, with asignal being generated and detected, as each nucleotide is incorporated.The benefits of such an approach are discussed in more detail inWO98/13523

[0052] Thus, the PPi detection enzymes (along with any enzyme substratesor other reagents necessary for the PPi detection reaction) may simplybe included in the polymerase reaction mixture.

[0053] More particularly, to carry out this embodiment of the method ofthe invention, the detection enzymes are included in the polymerasereaction step i.e. in the chain extension reaction step. Thus thedetection enzymes are added to the reaction mix for the polymerase stepprior to, simultaneously with or during the polymerase reaction. In thecase of an ELIDA detection reaction, the reaction mix for the polymerasereaction may thus include at least one nucleotide, polymerase,luciferin, APS, ATP suphurylase and luciferase. The polymerase reactionmay be initiated by addition of the polymerase or, more preferably thenucleotide, and preferably the detection enzymes are already present atthe time the reaction is initiated, or they may be added with thereagent that initiates the reaction. If a nucleotide degrading enzymesuch as apyrase is used, it will of course be understood that polymeraseaddition should not follow apyrase, if nucleotides are present.

[0054] The present invention thus permits PPi release to be detectedduring the polymerase reaction giving a real-time signal. The sequencingreactions may be continuously monitored in real-time.

[0055] The Rp isomer and/or the degradation products of NTPAS may beeliminated according to the present invention in a number of ways, andthe term “eliminated” requires simply that the Rp isomer and/ordegradation products be removed or excluded from the polymerase reactionstep. This may be achieved in any convenient way. For example, the Rpisomer may be excluded, by not being present in the NTPαS preparationwhich is added to or included in the polymerase reaction mixture.Alternatively, the NTPαS preparation may initially include the Rpisomer, but it may be removed, before, during or after, inclusion in oraddition to the polymerase reaction mixture, for example by enzymicdegradation. Combinations of means for eliminating the Rp isomer (e.g.removing and excluding) may also be used. Thus, the Rp isomer may beremoved or excluded from the polymerase reaction step, from the stepafter polymerisation has occurred (i.e. the nucleotide degradation step)or the detection of nucleotide incorporation step.

[0056] The degradation products of the NTPα-S may be removed during thepolymerisation step, the nucleotide degrading step or the detection ofnucleotide incorporation step. For example the degradation products areeliminated by enzymic degradation.

[0057] In one embodiment of the invention, the Rp isomer may thus beeliminated by using a preparation of NTPαS which contains only the Spisomer i.e. by using the Sp isomer of NTPαS only e.g. pure Sp isomer.Individual Rp and Sp isomers of dATPαS are available commercially (e.g.from Biolog Life Science, Bremen, Del.). Alternatively, such isomers mayreadily be synthesised stereo-specifically, or purified (separated) froma racemic mixture using techniques well known in the art and describedin the literature (see e.g. Eckstein, supra). dATPαS, along with theα-thio analogues of dCTP, dGTP and dTTP, may be purchased from AmershamPharmacia Biotech (Uppsala, SE).

[0058] The Rp isomer may be reduced in the reaction step to less than10% of the total dATPαS, preferably less than 5%, more preferably lessthan 3% Rp isomer.

[0059] In an alternative embodiment, the Rp isomer and/or degradationproducts of the α-thio nucleotide analogue may be removed by using anenzyme which degrades it, notably alkaline phosphatase. In particular,preliminary experimental results lead us to believe that alkalinephosphatase is capable of degrading both isomers of NTPαS, including theRp isomer and degradation products of NTPαS. Thus, Rp isomer anddegradation products of NTPαS present in the reaction mixture, or in theNTPαS preparation, may readily be removed (degraded) by adding analkaline phosphatase enzyme.

[0060] In a more particular embodiment, a combination of Sp isomertogether with an alkaline phosphatase enzyme may be used.

[0061] Conveniently, the alkaline phosphatase may simply be includedduring the polymerase reaction step. This may be achieved by adding theenzyme to the polymerase reaction mixture prior to, or simultaneouslywith, initiation of the polymerase reaction, or after the polymerasereaction has taken place, e.g. prior to adding nucleotides to thesample/primer/polymerase mixture to initiate the reaction, or after thepolymerase and nucleotide are added to the sample/primer mixture.

[0062] Alternatively, the alkaline phosphatase can be immobilized on asolid support e.g. a particulate solid support (e.g. magnetic beads) ora filter, or dipstick etc. and it may be added to the polymerasereaction mixture at a convenient time. When the Rp isomer has beendegraded, the immobilised enzyme may be removed from the reactionmixture (e.g. it may be withdrawn or captured, e.g. magnetically in thecase of magnetic beads), before the next nucleotide. is added. Theprocedure may then be repeated to sequence more bases.

[0063] Alkaline phosphatase catalyses the removal of 5′-phosphateresidues from nucleoside tri-, di- and mono-phosphates (includingribo-NTPs and dNTPs and, as mentioned above the both isomers of NTPαs).Thus, not only are nucleotides (nucleoside triphosphates) degraded butalso NDPs and NMPs. Shrimp, bacterial and/or calf-intestinal alkalinephosphatase are the preferred enzymes, but any suitable alkalinephosphatase enzyme may be used in the method of the invention, from anyconvenient source. The preferred enzyme is shrimp alkaline phosphatase.There are many commercial sources of alkaline phosphatase enzymes, orthey may, if desired, be isolated from a producing organism.

[0064] The amount of alkaline phosphatase enzyme to be used will dependupon the precise reaction system used, reaction conditions etc. and canreadily be determined by routine experiments. It has been found, forexample, that concentrations of 50 mU to 10 U may be employed.

[0065] As mentioned above, alkaline phosphatase has an activity indegrading NDPs and NMPs as well as NTPs. It further has the benefit ofacting on α-thio modified substrates also, thus including not onlyNTPαS, but also NDPαS and NMPαS. This activity lends a further benefitto the present invention. Thus, it has been observed that the enzymeapyrase, and also the enzyme luciferase used in the PPi detection methodpreferred according to the present invention, are inhibited when anNTPαS is used for nucleotide incorporation. The precise nature of theinhibitory effects observed is unclear, but may be due to NTPαS productor substrate inhibition. Thus, NTPαS may itself be inhibitory, or morelikely a degradation product of NTPαS (for example resulting fromapyrase action) is inhibitory. For example, it is postulated that thedegradation products NDPαS and NMPαS, (in particular dADPαS and dAMPαS)produced by the action of apyrase on NTPαS added to the polymerasereaction mixture for incorporation, may be inhibitory. Moreparticularly, it is believed that both the Sp and Rp isomers of NDPαSand/or NMPαS inhibit luciferase, and also the enzyme apyrase (seefurther in the Examples below). Such inhibitory substances may beremoved (or reduced) by the action of alkaline phosphatase.

[0066] As mentioned above, apyrase inhibition is indicated by a slowernucleotide degradation rate, and hence an increased signal width.Non-synchronised extension may also occur, and this limits the readlength attainable. Luciferase inhibition is indicated by a decrease insignal intensity. Thus, as shown further in the Examples below,inhibition of luciferase can be seen as a steady decrease in signal peakheight as the nucleotides are incorporated in the later cycles ofsequencing.

[0067] The effect of decreased signal intensity (luciferase inhibition)may combine with the effect of loss of signal definition (e.g. increasedsignal width or out of phase signals) (apyrase inhibition) to reduce theefficiency of the sequencing method, and hence its ability to providelonger read lengths. The use of alkaline phosphatase in conjunction withapyrase and dATPαS (and optionally other dNTPαS's) has the unexpectedadditional benefit of abrogating these significant additional sources ofinhibition (namely inhibition arising from the use of NTPαS togetherwith apyrase, e.g. due to NDPαS and/or NMPαS produced by apyraseaction).

[0068] Further benefits of using alkaline phosphatase may also beavailable. Thus, in certain conditions or circumstances, for examplelong-read sequencing (e.g. over 50 cycles), other, non-thio modified,NTPs added for incorporation, may also be a source of inhibition, albeitto a lesser extent than their α-thio modified analogues. For example,preliminary results suggests that dATP may be a source of inhibition forthe enzyme luciferase (see the Examples below). The action of apyrase onunincorporated NTPs results in the accumulation of degradation products,namely NDPs and NMPs (e.g. dADP, dAMP, dCTP, dCMP, dGDP, dGMP, dTDP,dTMP). In later cycles of sequencing, such products may inhibit enzymesused in the sequencing and/or PPi detection reactions e.g. apyraseand/or luciferase. Although any such inhibitory effects which may beobtained with other nucleotides will be lesser than that arising fromuse of NTPαS, they may still nonetheless contribute to a decreasedefficiency of the system. Again, any potential problem caused by suchinhibitory substances, may be removed or reduced by the action ofalkaline phosphatase.

[0069] ATP is generated in the first step of the ELIDA reaction(catalysed by the enzyme ATP Sulphurylase) used as the preferred methodfor PPi detection. Such an ATP product (in particular, any such ATP notused in the subsequent luciferase reaction) may also act as a substratefor apyrase (or other nucleotide degrading enzyme) and hence may alsolead to the generation of inhibitory products (e.g. ADP and AMP) whichmay inhibit luciferase, and to a lesser extent, also apyrase. Again, anysuch inhibitory products may be reduced or removed by the action ofalkaline phosphatase.

[0070] Finally, the use of alkaline phosphatase may further have abenefit in reducing any problems caused by kinase contamination. Kinasesmay be contaminants of enzyme preparations used in the sequencing andPPi detection reactions described herein. The action of kinases onapyrase degradation products (i.e. NDPs or NMPs), and ATP resulting fromthe ATP sulphurylase reaction in the ELIDA procedure may generate NTPswhich may distort the primary sequencing reaction (i.e may beincorporated by polymerase), and lead to non-synchronised extension.Alkaline phosphatase has a beneficial effect in degrading potentialkinase substrates.

[0071] The sample nucleic acid (i.e. the target nucleic acid to besequenced) may be any polynucleotide sequence it is desirable to obtainsequence information about. Thus, it may be any polynucleotide, orindeed oligonucleotide sequence. The nucleic acid may be DNA or RNA, andmay be natural or synthetic. Thus, the target nucleic acid may begenomic DNA, or cDNA, or a PCR product or other amplicon etc. The target(sample) nucleic acid may be used in any convenient form, according totechniques known in the art e.g. isolated, cloned, amplified etc., andmay be prepared for the sequencing reaction, as desired, according totechniques known in the art. The sample nucleic acid acts as a templatefor possible polymerase based extension of the primer and thus mayconveniently be referred to as “template” or “nucleic acid template”.The DNA may also be single or double-stranded—whilst a single-strandedDNA template has traditionally been used in sequencing reactions, orindeed in any primer-extension reaction, it is possible to use adouble-stranded template; strand displacement, or a localised opening-upof the two DNA strands may take place to allow primer hybridisation andpolymerase action to occur.

[0072] In the polymerase reaction, any convenient polymerase enzyme maybe used according to choice, as will be described in more detail below.In the case of a RNA template, such a polymerase enzyme may be a reversetranscriptase enzyme.

[0073] In order to repeat the method cyclically and thereby sequence thesample DNA and, also to aid separation of a single stranded sample DNAfrom its complementary strand, the sample DNA may optionally beimmobilised or provided with means for attachment to a solid support.

[0074] Moreover, the amount of DNA present in a sample to be analysedmay be small and it may therefore be desirable to amplify the DNA priorto sequencing. As mentioned above the sample DNA may thus be anamplicon.

[0075] Any desired method of in vitro or in vivo amplification may beused, e.g. PCR (or a variant or modification thereof) or Self SustainedSequence Replication (3SR) or the ligase chain reaction (LCR) etc.Whichever method of amplification is used, it may be convenient toimmobilise the amplified DNA, or provide it with means for attachment toa solid support. For example, a PCR primer may be immobilised or beprovided with means for attachment to a solid support.

[0076] Immobilisation of the amplified DNA may take place as part of PCRamplification itself, as where one or more primers are attached to asupport, or alternatively one or more of the PCR primers may carry afunctional group permitting subsequent immobilisation, e.g. a biotin orthiol group. Immobilisation by the 5′ end of a primer allows the strandof DNA emanating from that primer to be attached to a solid support andhave its 3′ end remote from the support and available for subsequenthybridisation with the extension primer and chain extension bypolymerase.

[0077] The solid support may conveniently take the form of microtitrewells. However, any solid support may conveniently be used, includingany of the vast number described in the art, e.g. forseparation/immobilisation reactions or solid phase assays. Thus, thesupport may also comprise particles (e.g. beads), fibres or capillariesmade, for example, of agarose, cellulose, alginate, Teflon orpolystyrene. Magnetic particles, e.g. the superparamagnetic beadsproduced by Dynal AS (Oslo, Norway) also may be used as a support.

[0078] The solid support may carry functional groups such as hydroxyl,carboxyl, aldehyde or amino groups, or other moieties such as avidin orstreptavidin, for the attachment of nucleic acid molecules e.g primers.These may in general be provided by treating the support to provide asurface coating of a polymer carrying one of such functional groups,e.g. polyurethane together with a polyglycol to provide hydroxyl groups,or a cellulose derivative to provide hydroxyl groups, a polymer orcopolymer of acrylic acid or methacrylic acid to provide carboxyl groupsor an aminoalkylated polymer to provide amino groups. U.S. Pat. No.4,654,267 describes the introduction of many such surface coatings.

[0079] If desired, the sample may be washed after a certain number ofreaction cycles e.g. 15-25, according to techniques well known in theart. Washing may be facilitated by immobilising the sample on a solidsurface. Using a nucleotide-degrading enzyme, combined with Rp isomerelimination (particularly using alkaline phosphatase) however, meansthat washing is not absolutely necessary.

[0080] The assay technique is very simple and rapid, thus making it easyto automate by using a robot apparatus where a large number of samplesmay be rapidly analysed. Since the preferred detection andquantification is based on a luminometric reaction, this can be easilyfollowed spectrophotometrically. The use of luminometers is well knownin the art and described in the literature.

[0081] The method of the present invention is particularly suited foruse in an array format, wherein samples are distributed over a surface,for example a microfabricated chip, and thereby an ordered set ofsamples may be immobilized in a 2-dimensional format. Many samples canthereby be analysed in parallel. Using the method of the invention, manyimmobilized templates may be analysed in this way by allowing thesolution containing the enzymes and one nucleotide to flow over thesurface and then detecting the signal produced for each sample. Thisprocedure can then be repeated. Alternatively, several differentoligonucleotides complementary to the template may be distributed overthe surface followed by hybridization of the template. Incorporation ofnucleotides may be monitored for each oligonucleotide by the signalproduced using the various oligonucleotides as primer. By combining thesignals from different areas of the surface, sequence-based analyses maybe performed by four cycles of polymerase reactions using the variousnucleotides.

[0082] The length of the extension primer is not critical and can beaccording to choice. It will be clear to persons skilled in the art thatthe size of the extension primer and the stability of hybridisation willbe dependent to some degree on the ratio of A-T to C-G base pairings,since more hydrogen bonding is available in a C-G pairing. Also, theskilled person will consider the degree of sequence identity between theextension primer to other parts of the template sequence and choose thedegree of stringency accordingly. Guidance for such routineexperimentation can be found in the literature, for example, MolecularCloning: a laboratory manual by Sambrook, J., Fritsch E. F. andManiatis, T. (1989). It may be advantageous to ensure that thesequencing primer hybridises at least one base inside from the 3′ end ofthe template to eliminate blunt-ended DNA polymerase activity. Ifseparate aliquots are used (i.e. 4 aliquots, one for each base), theextension primer is preferably added before the sample is divided intofour aliquots although it may be added separately to each aliquot.

[0083] Alternatively, a primer with a phosphorylated 5′-end, containinga loop and annealing back on itself and the 3′-end of the singlestranded template can be used. If the 3′-end of the template has thesequence region denoted T (template), the primer has the followingsequence starting from the 5′-end; P-L-P′-T′, where P is primer specific(5 to 30 nucleotides), L is loop (preferably 4 to 10 nucleotides), P′ iscomplementary to P (preferably 5 and 30 nucleotides) and T′ iscomplementary to the template sequence in the 3′-end (T) (at least 4nucleotides). This primer can then be ligated to the single strandedtemplate using T4 DNA ligase or a similar enzyme. This provides acovalent link between the template and the primer, thus avoiding thepossibility that the hybridised primer is washed away during theprotocol.

[0084] In the polymerase reaction, any convenient polymerase enzyme maybe used according to choice, e.g. T7 polymerase, Klenow or SequenaseVer. 2.0 (USB U.S.A.). Any suitable polymerase may conveniently be usedand many are known in the art and reported in the literature. However,it is known that many polymerases have a proof-reading or error checkingability and that 3′ ends available for chain extension are sometimesdigested by one or more nucleotides. If such digestion occurs in themethod according to the invention the level of background noiseincreases. In order to avoid this problem, a nonproof-readingpolymerase, e.g. exonuclease deficient (exo⁻) Klenow polymerase may beused and this is preferred according to the present invention.Alternatively, substances which suppress 3′ digestion by polymerase,such as fluoride ions or nucleotide monophosphates, may be used. Theprecise reaction conditions, concentrations of reactants etc. mayreadily be determined for each system according to choice. However, itmay be advantageous to use an excess of polymerase over primer/templateto ensure that all free 3′ ends are extended.

[0085] In the method of the invention, it is preferred to use a DNApolymerase with high efficiency in each extension step due to the rapidincrease of background signal which may take place if templates whichare not fully extended accumulate. A high fidelity in each step is alsodesired, which can be achieved by using polymerases with exonucleaseactivity. However, this has the disadvantage mentioned above thatdegradation of the extending strand may occur. Although the exonucleaseactivity of the Klenow polymerase is low, the 3′ end of the primer maybe degraded with longer incubations in the absence of nucleotides. Aninduced-fit binding mechanism in the polymerisation step selects veryefficiently for binding of the correct dNTP with a net contributiontowards fidelity of 10⁵-10⁶. Exonuclease-deficient polymerases, such as(exo⁻) Klenow or Sequenase 2.0, catalysed incorporation of a nucleotidewhich was only observed when the complementary dNTP was present,confirming a high fidelity of these enzymes even in the absence ofproof-reading exonuclease activity. The main advantage of using (exo⁻)Klenow DNA polymerase over Sequenase 2.0 is its lower Km fornucleotides, allowing a high rate of nucleotide incorporation even atlow nucleotide concentrations. As mentioned above, it is also possibleto replace all dNTPs with nucleotide analogues or non-naturalnucleotides such as dNTPαS, and such analogues may be preferable for usewith a DNA polymerase having exonuclease activity.

[0086] In certain circumstances, e.g. with longer sample templates, itmay be advantageous to use a polymerase which has a lower K_(M) forincorporation of the correct (matched) nucleotide, than for theincorrect (mismatched) nucleotide. This may improve the accuracy andefficiency of the method. Suitable such polymerase enzymes include theα-polymerase of Drosophila.

[0087] In many diagnostic applications, for example genetic testing forcarriers of inherited disease, the sample will contain heterozygousmaterial, that is half the DNA will have one nucleotide at the targetposition and the other half will have another nucleotide. Thus if fouraliquots (i.e. four parallel reactions of the same sample) are used inan embodiment according to the invention, two will show a negativesignal and two will show half the positive signal. It will be seentherefore that it is desirable to quantitatively determine the amount ofsignal detected in each sample. Also, it will be appreciated that if twoor more of the same base are adjacent the 3′-end of the primer a largersignal will be produced. In the case of a homozygous sample it will beclear that there will be three negative and one positive signal when thesample is divided into four parallel reactions.

[0088] In carrying out the method of the invention, any possiblecontamination of the reagents e.g. the NTP solutions, by PPi isundesirable and may readily be avoided by including a pyrophosphatase,preferably in low amounts, in the reagent solutions. Indeed, it isdesirable to avoid contamination of any sort and the use of high purityor carefully purified reagents is preferred, e.g. to avoid contaminationby kinases.

[0089] Reaction efficiency may be improved by including Mg²⁺ ions in thereagent (NTP and/or polymerase) solutions.

[0090] A potential problem which has previously been observed withPPi-based sequencing method arises when the DNA to be sequenced has anumber of identical adjacent bases, especially three or more of thesame. Further, false signals may occur due to mispriming, i.e.hybridisation of the primer not to its targeted complement within thetarget DNA sequence but to another region, which will result ingeneration of “incorporation signals” which do not reflect the identityof the target sequence. As described in WO00/43540, this may be avoidedby the use of single-stranded nucleic acid binding proteins in thereaction mixture after annealing of the primer to the template.

[0091] Thus, a further preferred feature of the invention is the use ofa single-stranded nucleic acid binding protein, which is included duringthe polymerase reaction step after primer annealing.

[0092] As mentioned above, the benefits of the present invention arisefrom the elimination (e.g. removal and/or exclusion) of inhibitingsubstances from the reaction mixture.

[0093] Thus, viewed from a different aspect, the present inventionprovides a method of decreasing the inhibition of polymerase in aPPi-based sequencing procedure which uses at least one NTPαS, saidmethod comprising eliminating the Rp isomer of NTPαS and/or thedegradation products of said NTPαS from the sequencing reaction mixture(i.e. the template, primer, polymerase and/or nucleotide mix).

[0094] Further, since a similar inhibiting effect has been observed forthe apyrase enzyme, which may be used to degrade un-incorporatednucleotides, the present invention also provides a method of decreasingthe inhibition of apyrase when used in a PPi-based sequencing procedurewhich uses at least one NTPαS, said method comprising eliminating the Rpisomer of NTPαS and/or the degradation products of said NTPαS from thesequencing reaction.

[0095] As further mentioned above, the luciferase enzyme which ispreferably used in PPi detection may be inhibited by various inhibitorysubstances (e.g. Rp isomer and/or degradation products) and these mayadvantageously be removed by the action of alkaline phosphatase.Accordingly, in a further aspect the present invention provides a methodof decreasing the inhibition of luciferase when used as a detectionenzyme in a PPi-based sequencing procedure, said method comprisingincluding alkaline phosphatase in the sequencing and/or PPi detectionreaction mixture.

[0096] Typically, in certain embodiments of the invention NTPαS will beATPαS (e.g. dATPαS or ddATPαS).

[0097] The invention also comprises kits for use in methods of theinvention which will normally include at least the following components:

[0098] (a) a polymerase;

[0099] (b) means for detecting pyrophosphate release (preferably enzymemeans, and most preferably luciferase and ATP sulphurylase, e.g. thereaction components of an ELIDA assay (see above);

[0100] (c) optionally a nucleotide-degrading enzyme (preferablyapyrase);

[0101] (d) alkaline phosphatase

[0102] (e) one or more nucleotides, preferably deoxynucleotides,including, in place of an adenine nucleotide (e.g. dATP), anα-thiotriphoshate analogue of said nucleotide (e.g. dATPαS);

[0103] (f) optionally, a test specific primer which hybridises to samplenucleic acid so that the target position is in close proximity to the 3′end of the primer;

[0104] A further embodiment of the kit of the invention will normallyinclude at least the following components:

[0105] (a) a polymerase;

[0106] (b) means for detecting pyrophosphate release (preferably enzymemeans, and most preferably luciferase and ATP sulphurylase, e.g. thereaction components of an ELIDA assay (see above);

[0107] (c) optionally a nucleotide-degrading enzyme (preferablyapyrase);

[0108] (d) the Sp isomer of an α-thiotriphosphate analogue of an adeninenucleotide (preferably dATPαS) and optionally one or more Sp isomers ofthymine, cytosine or guanine nucleotides (preferably dGTPαS, dCTPαS ordGTPα); and

[0109] (f) optionally, a test specific primer which hybridises to samplenucleic acid DNA so that the target position is in close proximity tothe 3′ end of the primer;

[0110] (g) optionally, one or more unmodified thymine, cytosine orguanine nucleotides (preferably dTTP, dCTP, dGTP);

[0111] (h) optionally, alkaline phosphatase.

[0112] The benefits of the present invention in reducing the effects ofinhibitory substances in PPi-based sequencing, improve the efficiencyand reliability of the method and extend the applications in which themethod can be used. Thus, techniques requiring longer reads such asgenome re-sequencing, comparative EST sequencing, microbial typing andconfirmatory sequencing may all be conducted by PPi-based sequencingaccording to the present invention.

[0113] The primer, if present in the kits, is designed so that it bindsto the template nucleic acid with its 3′ end in close proximity to thetarget nucleotide. By close proximity it is meant 1 to 30 nucleotides,preferably 1 to 20 nucleotides, more preferably 1 to 10, most preferably1 to 5 nucleotides from the target nucleotide.

[0114] The invention will now be described by way of non-limitingexamples with reference to the drawings in which:

[0115]FIG. 1 shows a trace (light intensity v nucleotide addition)obtained from simulation of the effect of dATP on luciferase andapyrase, using 2 μM ATP in the nucleotide solution. In this experiment,100 ng luciferase, 50 mU apyrase, 0.1 M Tris-acetate (pH 7.75), 0.5 mMEDTA, 5 mM magnesium acetate, 0.1% bovine serum albumin, 1 mMdithiothreitol, 0.4 mg/ml polyvinylpyrrolidone (360 000), and 100 μg/mlD-luciferin (BioThema) were used. 200 μl of 0.7 mM nucleotide (dATP)containing 2 μM ATP is dispensed into the reaction mixture as describedin Example 1. The height of the ascending curve demonstrates luciferaseactivity and the slope of the descending curve demonstrates the apyraseactivity. The PPi was detected in real time.

[0116]FIG. 2 shows a trace (light intensity v nucleotide addition)obtained from simulation of the effect of dATPαS on luciferase andapyrase, using 2 μM ATP in the nucleotide solution. In this experiment,100 ng luciferase, 50 mU apyrase, 0.1 M Tris-acetate (pH 7.75), 0.5 mMEDTA, 5 mM magnesium acetate, 0.1% bovine serum albumin, 1 mMdithiothreitol, 0.4 mg/ml polyvinylpyrrolidone (360 000), and 100 μg/mlD-luciferin (BioThema) were used. 200 μl of 0.7 mM nucleotide (dATPαS)containing 2 μM ATP is dispensed to the reaction mixture as described inExample 1. The height of ascending curve demonstrates luciferaseactivity and the slope of descending curve demonstrates the apyraseactivity.

[0117]FIG. 3 shows a trace (light intensity v nucleotide addition)obtained from simulation of the effect of dCTP on luciferase andapyrase, using 2 μM ATP in the nucleotide solution. In this experiment,100 ng luciferase, 50 mU apyrase, 0.1 M Tris-acetate (pH 7.75), 0.5 mMEDTA, 5 mM magnesium acetate, 0.1% bovine serum albumin, 1 mMdithiothreitol, 0.4 mg/ml polyvinylpyrrolidone (360 000), and 100 μg/mlD-luciferin (BioThema) were used. 200 μl of 0.2 mM nucleotide (dCTP)containing 2 μM ATP is dispensed to the reaction mixture as described inExample 1. The height of ascending curve demonstrates luciferaseactivity and the slope of descending curve demonstrates the apyraseactivity.

[0118]FIG. 4 shows a trace (light intensity v nucleotide addition)obtained from simulation of the effect of dGTP on luciferase andapyrase, using 2 μM ATP in the nucleotide solution. In this experiment,100 ng luciferase, 50 mU apyrase, 0.1 M Tris-acetate (pH 7.75), 0.5 mMEDTA, 5 mM magnesium acetate, 0.1% bovine serum albumin, 1 mMdithiothreitol, 0.4 mg/ml polyvinylpyrrolidone (360 000), and 100 μ/mlD-luciferin (BioThema) were used. 200 μl of 0.16 mM nucleotide (dGTP)containing 2 μM ATP is dispensed to the reaction mixture as described inExample 1. The height of ascending curve demonstrates luciferaseactivity and the slope of descending curve demonstrates the apyraseactivity.

[0119]FIG. 5 shows a trace (light intensity v nucleotide addition)obtained from simulation of the effect of dTTP on luciferase andapyrase, using 2 μM ATP in the nucleotide solution. In this experiment,100 ng luciferase, 50 mU apyrase, 0.1 M Tris-acetate (pH 7.75), 0.5 mMEDTA, 5 mM magnesium acetate, 0.1% bovine serum albumin, 1 mMdithiothreitol, 0.4 mg/ml polyvinylpyrrolidone (360 000), and 100 μg/mlD-luciferin (BioThema) were used. 200 μl of 0.8 mM nucleotide (dTTP)containing 2 μM ATP is dispensed to the reaction mixture as described inExample 1. The height of ascending curve demonstrates luciferaseactivity and the slope of descending curve demonstrates the apyraseactivity.

[0120]FIG. 6 shows a trace (light intensity v nucleotide addition)obtained from simulation of the effect of dATPαS on luciferase andapyrase, using 2 μM ATP in the nucleotide solution, in the presence ofalkaline phosphatase. In this experiment, 100 ng luciferase, 50 mUapyrase, 0.1 M Tris-acetate (pH 7.75), 0.5 mM EDTA, 5 mM magnesiumacetate, 0.1% bovine serum albumin, 1 mM dithiothreitol,. 0.4 mg/mlpolyvinylpyrrolidone (360 000), 100 μg/ml D-luciferin (BioThema), and 1Ualkaline phosphatase were used. 200 μl of 0.7 mM nucleotide (dATPαS)containing 2 μM ATP is dispensed to the reaction mixture as described inExample 1. The height of ascending curve demonstrates luciferaseactivity and the slope of descending curve demonstrates the apyraseactivity.

[0121]FIG. 7 shows three traces (light intensity v nucleotide addition)obtained in DNA sequencing reactions using 1 pmol of primedoligonucleotide template; the reaction system contained 10 U Klenow DNApolymerase, 25 mU ATP sulphurylase and 0.1 M Tris-acetate (pH 7.75), 0.5mM EDTA, 5 mM magnesium acetate, 0.1% bovine serum albumin, 1 mMdithiothreitol, 0.4 mg/ml polyvinylpyrrolidone (360 000), and 100 μg/mlD-luciferin (BioThema); and A) 100 ng luciferase and 50 mU apyrase; B)100 ng luciferase, 50 mU apyrase and 2 mU nucleoside diphosphate kinase;C) 100 ng luciferase, 50 mU apyrase, 2 mU nucleoside diphosphate kinaseand 2 U alkaline phosphatase. The arrows on B indicates the falsesignals appearing as a result of kinase activity in the system, whichare removed by addition of alkaline phosphatase in the experiment shownin C.

[0122]FIG. 8 presents traces (light intensity v nucleotide addition)showing the effects of pre-incubating a PPi-sequencing reaction mixture(in the absence of template) with varying amounts of “normal” (i.e.racemic) dATPαS (containing both Rp and Sp isomers) ((A) 0 μl; (B) 5 μl;(C) 10 μl; (D) 20 μl) or the pure Sp isomer of dATPαS ((E) 0 μl; (F) 5μl; (G) 10 μl; (H) 20 μl) or the pure Rp isomer of dATPαS ((I) 0 μl; (J)5 μl; (K) 10 μl; (L) 20 μl). After pre-incubation, template was addedand the PPi-based sequencing reaction was performed as described inExample 2.

[0123]FIG. 9 is a trace (light intensity v nucleotide addition) showingthe results of a DNA sequencing reaction on a pUC19-derived templatewherein the four different nucleotides are added stepwise to thetemplate hybridized to a primer. A mixture of the Rp and Sp isomers ofdATPαS is used. The template/primer was incubated with 10U (exo⁻) Klenowand 40 mU apyrase, and other components of the sequencing/PPi detectionreaction as described in Example 3. The reaction was started by theaddition of the first nucleotide, and the nucleotides were added in astepwise fashion. The PPi released was detected in real time byluciferase.

[0124]FIG. 10 is a trace (light intensity v nucleotide addition) showingthe results of a DNA sequencing reaction on a pUC19-derived templatewherein the four nucleotides are added stepwise to the templatehybridized to the primer. Only the pure Sp isomer of d-ATPαS was used.The template/primer was incubated with 10U (exo⁻) Klenow and 40 MUapyrase and other components of the sequencing/PPi detection reaction asdescribed in Example 3. The reaction was started by the addition of thefirst nucleotide, and nucleotides were then added in a stepwise fashion.The PPi released was detected in real time by luciferase.

EXAMPLE 1 Investigation of the Inhibitory Effects of DifferentNucleotides in PPi-Based Sequencing Using an ELIDA DetectionReaction—the Effects of Alkaline Phosphatase

[0125] In this Example a series of 5 experiments were carried out toexamine the inhibitory effect of each of the 5 nucleotides (dATP,dATPαS, dCTP, dGTP and dTTP) on the enzymes apyrase and luciferase. Amodel system was used which simulated the effects of these enzymes in aPPi-based sequencing reaction. Each nucleotide was added to theluciferase/apyrase/substrate mixture, along with ATP (to provide asubstrate for luciferase), and the effect of each nucleotide addition onthe enzymes was monitored by observing the light signals generated fromthe luciferase reaction. A further two experiments demonstrate, firstly,that the inhibitory effects resulting from use of dATPαS may beabrogated in the model system by the use of alkaline phosphatase, and,secondly that alkaline phosphatase further has an additional benefit incounteracting the interfering effects of kinase enzymes on the reactionsystem (as demonstrated in a sequencing reaction).

[0126] The reactions were performed at room temperature in a volume of50 μl on an automated prototype Pyrosequencer instrument (kindlysupplied by Pyrosequencing AB, Uppsala, Sweden). The reaction mixturecontained: 50 mU apyrase (Sigma Chemical Co., USA), 100 ng purifiedluciferase (BioThema, Dalarö, Sweden), 0.1 M Tris-acetate (pH 7.75), 0.5mM EDTA, 5 mM magnesium acetate, 0.1% bovine serum albumin, 1 mMdithiothreitol, 0.4 mg/ml polyvinylpyrrolidone (360 000), and 100 μg/mlD-luciferin (BioThema). An ATP concentration of 2 μM was added todifferent nucleotides (dATP, dATPαS, dCTP, dGTP and dTTP), as shown inFIGS. 1 to 5 to follow the activity of luciferase and apyrase. Forinvestigation of the effect of Shrimp Alkaline Phosphatase (AmershamParmacia Biotech, Uppsala, Sweden), two units of this enzyme were addedto the reaction mixture—see further below. In total, 50 additions ofnucleotide and ATP mixture were performed in 50 minutes. The output oflight resulting from nucleotide incorporation was detected by aCCD-camera. The data was obtained in Excel format.

[0127] Seven experiments were performed. Experiments 1 to 5 investigatedthe effects of each of dATP, dATPαS, dCTP, dGTP and dTTP. Experiment 1was peformed using an admixture of 0.7 mM nucleotide dATP and 2 μM ATPdispensed to the reaction mixture. The trace obtained from experiment 1is presented as FIG. 1. The height of the ascending curve demonstratesluciferase activity and the slope of the descending curve is indicativeof or demonstrates apyrase activity. Note the relatively linear decreasein signal intensity, which is due to inhibition of luciferase, believedto be due to accumulation of degradation products of dATP (e.g. dAMP).It has earlier been reported that dAMP inhibits luciferase (Ford et al.,1998, Methods in Mol. Biol., 102: 3-20) and accordingly it is inferredfrom this that the inhibitory substance may be dAMP. Thus, luciferaseinhibition is shown, when dATP is used, and this is believed to be dueto the degradation products of dAMP (dATP and/or dADP).

[0128] Experiment 2 was performed using an admixture of 0.7 mMnucleotide dATPαS and 2 μM ATP dispensed to the reaction mixture. Theresults are shown in FIG. 2. The height of the ascending curvedemonstrates luciferase activity and the slope of the descending curvedemonstrates apyrase activity. Note the relatively linear decrease insignal intensity, which is due to inhibition of luciferase, believed tobe caused by accumulation of inhibitory products (e.g. dAMPαS). Thedecrease in signal intensity after 50 cycles is 50% less than the dataobtained from degradation of dATP in FIG. 1. Accordingly, it is inferredfrom this that just one isomer of dATPαS is inhibitory, and this isbelieved to be the Sp-isomer (or rather that the products of Sp isomerare inhibitory to luciferase). Apyrase is drastically inhibited in latercycles as seen by the wider peaks which are obtained. The inhibitoryeffect after each addition is believed to be due to accumulation of theRp isomer of dATPαS, and also the product(s) of the degradation of theSp isomer. It is believed that apyrase degrades only the Sp isomer andnot the Rp isomer. It is speculated that the inactive isomer of dATPαS(Rp) is not recognised by luciferase since it is not degraded to formthe inhibitory product.

[0129] Experiment 3 was performed using an admixture of 0.2 mMnucleotide dCTP and 2 μM ATP dispensed to the reaction mixture. Theresults are shown in FIG. 3. The height of the ascending curvedemonstrates luciferase activity and the slope of the descending curvedemonstrates apyrase activity. Note the relatively constant signalintensity even after 50 cycles which indicates that the products of thisnucleotide do not inhibit luciferase or apyrase under the conditionsused, and within the number of cycles shown.

[0130] Experiment 4 was performed using an admixture of 0.16 mMnucleotide dGTP and 2 μM ATP dispensed to the reaction. The resultsobtained from experiment 4 are presented in FIG. 4. The height of theascending curve demonstrates luciferase activity and the slope of thedescending curve demonstrates the apyrase activity. Note the relativelyconstant signal intensity even after 50 cycles which indicate that theproducts of this nucleotide do not inhibit luciferase or apyrase underthe conditions used, and within the number of cycles shown.

[0131] Experiment 5 was performed using an admixture of 0.8 mMnucleotide dTTP and 2 μM ATP dispensed to the reaction. The results areshown in FIG. 5. The height of ascending curve demonstrates luciferaseactivity and the slope of descending curve demonstrates the apyraseactivity. Note the relatively constant signal intensity even after 50cycles which indicate that the products of this nucleotide do notinhibit luciferase or apyrase, under the conditions used, and within thenumber of cycles shown.

[0132] Experiment 6 investigated the reduction in inhibition effected bythe addition of alkaline phosphatase to the reaction mixture. Thisexperiment was performed similarly as for Experiment 2, using anadmixture of 0.7 mM nucleotide dATPαS and 2 μM ATP dispensed to thereaction mixture. The results are shown in FIG. 6. The height of thegenerated peak demonstrates luciferase activity and the slope of thedescending curve demonstrates the apyrase activity. Note the efficiencyof nucleotide degradation as compared with FIG. 2. It can be seen thatthe signal width remains constant even after 50 cycles indicating lackof apyrase inhibition, and hence the high efficiency of alkalinephosphatase in preventing this inhibition (believed to be due to itsaction in degrading both isomers (i.e. the Rp and Sp isomers) ofdATPαS). No decrease in signal intensity is observed, indicating noinhibition of luciferase. This is believed to be due to the effect ofalkaline phosphatase in degrading the products of dATPαS (thus removingthe inhibitory elements). The experimental conditions for FIGS. 2 and 6are the same except for addition of 2 U AP in the reaction mixture inExperiment 6. Thus, the results of Experiment 6 show that alkalinephosphatase can remove inhibitory substances from the reaction system,thus improving the performance of the luciferase and apyrase enzymes.

[0133] Experiment 7 investigated the inhibitory effects of nucleotidesand kinases upon a sequencing reaction, and the reduction of theseeffects using alkaline phosphatase. For investigation of the effect ofkinase, 2 mU of kinase (Sigma Chemicals) was added to the sequencingreaction mixture containing: 0.5 pmol primed synthetic templateE3PN/NUSPT (Ronaghi et al. Science, 1998), 10 U exonuclease-deficientKlenow DNA polymerase (Amersham Pharmacia Biotech, Uppsala, Sweden), 40mU apyrase (Sigma Chemical Co., USA), 100 ng purified luciferase(BioThema, Dalaro, Sweden), 15 mU of recombinant produced ATPsulfurylase, 0.1 M Tris-acetate (pH 7.75), 0.5 mM EDTA, 5 mM magnesiumacetate, 0.1% bovine serum albumin, 1 mM dithiothreitol, 5 μM adenosine5-phosphosulfate (APS), 0.4 mg/ml polyvinylpyrrolidone (360 000), and100 μg/ml D-luciferin (BioThema). The sequencing procedure was carriedout by stepwise elongation of the primer-strand upon sequential additionof Sp-dATPαS (Biolog Life Science, Bremen, Del.), dCTP, dGTP, and dTTP(Amersham Pharmacia Biotech) and simultaneous degradation of nucleotidesby apyrase. To study the effect of alkaline phosphatase on kinaseactivity, 2 mU of kinase enzyme and 2 U of alkaline phosphatase wereadded to the above admixture. The output of light resulting fromnucleotide incorporation was detected by a CCD-camera. The data wasobtained in Excel format. FIG. 7a shows the results of the sequencingreaction in the absence of added kinase or alkaline phosphatase. FIG. 7bshows a trace of the results of the sequencing reaction in the presenceof 2 mU of nucleoside diphosphate kinase. The arrow indicates the falsesignals that are generated.

[0134] Thus, it can be seen that the addition of alkaline phosphatase tothe polymerisation reaction mixture removes the false signals generatedby kinase contamination, by removing the substrates for the kinase fromsolution.

EXAMPLE 2 Inhibitory Efects of dATPαS on PPi-Based Sequencing

[0135] In this example, the inhibitory effect of dATPαS on a PPi-basedsequencing reaction with ELIDA detection (known as Pyrosequencing™) wasinvestigated by preincubating the reaction mixture in the absence oftemplate with varying amounts of the normal (i.e. racemic) dATPαS(containing both Rp and Sp isomers), or the pure Sp isomer of dATPαS.After preincubation, template was added and a normal “Pyrosequencing™”,reaction was carried out.

[0136] Materials and Methods

[0137] The “normal” dATPαS was taken from the PSQ 96 SNP Reagent kitsupplied by Pyrosequencing AB (Uppsala, SE). The pure Rp and Sp isomerswere purchased from Biolog Life Science (Bremen, Germany). The reactionmixture also included Enzyme mix (DNA polymerase, ATP-sulphurylase,apyrase and luciferase) and Substrate mix (luciferin and APS), from thePSQ 96 SNP Reagent kit supplied by Pyrosequencing AB. The template wasan oligonucleotide (interactive, Ulm, Germany) from which the followingsequence could be read after annealing of a sequencing primer:CTAAAGGTGCACCATGACTGGGGTTACAGTCATC.

[0138] The pure isomer samples were diluted to the same concentration asthe dATPαS in the PSQ 96 SNP Reagent kit. For preincubation, 0, 5, 10 or20 μl of each A-sample (normal dATPαS or the Rp or Sp isomer) was addedto a mixture containing 5 μl Enzyme mix, 5 μl Substrate mix in a totalvolume of 45 μl. The reaction was carried out in the 96 well platedelivered with the PSQ 96 SNP Reagent kit. The reaction was incubated atroom temperature, in the dark for 10 minutes. After this, 1.5 pmol in 5μl annealed oligonucleotide template was added to each well and theplate was transferred from the PSQ 96 instrument, where a normal“Pyrosequencing™” reaction was carried out (omitting addition of Enzymeand Substrate mixes).

[0139] The results are shown in FIG. 8. Using normal (i.e. racemic)dATPαS, it can be seen that pre-incubation with 5 μl (FIG. 8B) 10 μl(FIG. 8C) or 20 μl (FIG. 8D) results in progressively severe inhibitionof the sequencing reaction (as compared with 0 μl of dATPαS added (FIG.8A)). Some loss of signal intensity (peak height) and moresignificantly, loss of signal definition (e.g. broader, less defined andless clean signals) can be seen. Thus, it can be seen that apyraseinhibition is particularly occurring. Even more pronounced inhibition isseen when the Rp isomer is used (see FIG. 8J). This effect wassignificantly reduced when using the pure Sp isomer in place of racemicdATPαS or Rp isomer of dATPαS, as seen from FIG. 8F (5 μl), FIG. 8G (10μl) and FIG. 8H (20 μl) (compared to 0 μl (FIG. 8E)). The pure Rp isomerhas a severe effect on all enzymes involved in the ELIDA detection(FIGS. 8I and 8J). Apyrase inhibition is indicated by increased signalwidth. This can clearly be seen in FIGS. 8J to 8L, most clearly it isdemonstrated on FIG. 8K. Luciferase inhibition is indicated by decreasedsignal intensity. As can be seen on FIG. 8K, the signal intensity isdecreasedin comparison with FIG. 8I, indicating that luciferase is beinginhibited.

[0140] A similar experiment was also performed where the preincubationwas carried out comparing “normal” dATPαS with dCTP, dGTP or dTTP, allfrom a prototype to the PSQ 96 SNP Reagent kit. As control, only 1×TE(dilution buffer for the dNTPs) was added. The template was the same asabove, but 2 pmol were added to each well.

[0141] The results obtained (not shown) clearly showed no negativeeffect from preincubation with dCTP, dGTP or dTTP, but confirmed asevere negative effect from dATPαS, mainly on apyrase.

EXAMPLE 3 PPi-Based DNA Sequencing With and Without Rp-dATPαS

[0142] In this Example, two sequencing experiments were performed usingthe same template (a PCR product of the standard cloning plasmid pUC19),with and without the Rp isomer of dATPαS (i.e. using firstly a racemicmixture of dATPαS, and secondly pure Sp-dATPαS).

[0143] Single Stranded Template Preparation

[0144] The standard plasmid pUC19 was used to generate the template byPCR. In brief, PCR primers GGGATCATGTAACTCGCCTTGA (Upper primer,biotinylated position 1345) and CGGGAGGGCTTACCATCTGG (lower primer,position 1648), (where positions 1648−1345=303 bp) were used in a PCRreaction on pUC19 to generate a fragment of 303 bp in length. Fiftymicroliters of biotinylated PCR product was immobilized onto 20 μlstreptavidin-coated super paramagnetic beads (DynabeadsM-280-streptavidin, Dynal AS, Oslo, Norway) by incubation at 43° C. for30 minutes. Single-stranded DNA was obtained by incubating theimmobilized PCR product in 5 μl 0.1 M NaOH for 4 minutes. Theimmobilized strand was resolved in 8 μl H₂O plus 1 μl annealing buffer(100 mM Tris-Ac₂ (pH 7.75), 20 mM MgAc₂). Single-stranded DNAcorresponding to 50 μl PCR product was hybridized to 10 pmol sequencingprimer (TCAGCAATAAACCAGCCAGCC) at 70° C. for 3 minutes followed byincubation at room temperature for 5 minutes. (After annealing of thesequencing primer, the length of single-stranded template remaining is211 bp.) The primed PCR product was added to the Pyrosequencing™reaction mixture containing: 0.1 M Tris-Ac₂ (pH 7.75), 0.05% Tween 20,10 U exonuclease deficient (exo⁻) Klenow DNA polymerase, 40 mU apyrase(Sigma Chemical Co. St. Louis, Mo., USA), 0.8 μg purified luciferase(BioThema, Dalarö, Sweden), 15 mU recombinant ATP sulfurylase(Karamohamed et al., 1999), 0.5 μg single-stranded DNA binding protein(Amersham Pharmacia Biotech., Uppsala, Sweden), 0.5 mM EDTA, 5 mM MgAc₂,0.1% bovine serum albumin (BioThema), 1 mM dithiothreitol, 5 μMadenosine 5′-phosphosulfate (Sigma Chem. Co.), 0.4 mg/mlpolyvinylpyrrolidone (360 000), and 100 μg/ml D-luciferin (BioThema) ina total volume of 50 μl.

[0145] Pyrosecuencing™

[0146] Pyrosequencing™ was performed at room temperature on an automatedPyrosequencer prototype model (Pyrosequencing AB, Uppsala, Sweden;www.pyrosequencing.com) at a dispensing pressure of 600 mbar with 8 msecopen time and 60 sec cycle time. The sequencing procedure was carriedout by stepwise elongation of the primer-strand upon cyclic dispensationof the different deoxynucleoside triphosphates (Amersham PharmaciaBiotech). In one experiment (shown in FIG. 9), a racemic mixture ofdATPαS (“normal” dATPαS) is used, (along with dCTP, dGTP and dTTP) andin the second experiment pure Sp isomer of dATPαS is used. The output oflight resulting from nucleotide incorporation was detected by aphotomultiplier. The data was obtained in Microsoft Excel and is shownin FIGS. 9 and 10.

[0147] Looking at FIG. 9 it can be seen that signal quality graduallydeteriorates with repeated nucleotide addition, leading eventually toloss of readable signal (parts 4-6 of FIG. 9). In FIG. 10, it will beseen that with pure Sp isomer, signal quality is maintained for longer,and a longer read-length is obtained before signal quality deterioratesbelow readable.

1. A method of identifying a base at a target position in a samplenucleic acid sequence, said method comprising: subjecting a primerhybridised to said sample nucleic acid immediately adjacent to thetarget position, to a polymerase primer extension reaction in thepresence of a nucleotide, whereby the nucleotide will only becomeincorporated if it is complementary to the base in the target position,and determining whether or not said nucleotide is incorporated bydetecting whether PPi is released, the identity of the target base beingdetermined from the identity of any nucleotide incorporated, wherein,where said nucleotide comprises an adenine base, an α-thio triphosphateanalogue of said nucleotide is used, and the Rp isomer of said analogueand/or the degradation products of said analogue are eliminated from thepolymerase reaction step.
 2. A method as claimed in claim 1 wherein theRp isomer is eliminated by using a preparation of an α-thio triphosphateanalogue of an adenine nucleotide which contains only the Sp isomerthereof.
 3. A method as claimed in claim 2 wherein the Rp isomer iseliminated by using a preparation of dATPαS or dd ATPαS which containsonly the Sp isomer thereof.
 4. A method as claimed in claim 1 whereinthe Rp isomer and/or the degradation products of said analogue areeliminated by enzymic degradation.
 5. A method as claimed in claim 4wherein alkaline phosphatase is included in or added to the polymerasereaction mixture.
 6. A method as claimed in any preceding claim whereina nucleotide degrading enzyme is present during or after the polymerasereaction step.
 7. A method as claimed in claim 6 wherein the nucleotidedegrading enzyme is apyrase.
 8. A method as claimed in claim 2 whereinalkaline phosphatase is included in or added to the polymerase reactionmixture.
 9. A method as claimed in any one of claims 1 to 8 wherein theprimer extension reaction is repeated in the presence of furthernucleotides.
 10. A method of decreasing the inhibition of apyrase whenused in a PPi-based sequencing procedure which uses at least one NTPαS,said method comprising eliminating the Rp isomer of NTPαS and/or thedegradation products of said analogue from the sequencing reaction. 11.A method of decreasing the inhibition of luciferase when used in aPPi-based sequencing procedure which uses at least one NTPαS, saidmethod comprising eliminating the Rp isomer of NTPαS and/or thedegradation products of said analogue from the sequencing reaction. 12.A kit for use in a method of identifying a base at a target position ina nucleic acid which comprises: (a) a polymerase; (b) means fordetecting pyrophosphate release; (c) optionally a nucleotide-degradingenzyme; (d) alkaline phosphatase (e) one or more nucleotides, preferablydeoxynucleotides, including, in place of an adenine nucleotide, anα-thiotriphoshate analogue of said nucleotide; (f) optionally, a testspecific primer which hybridises to sample nucleic acid so that thetarget position is in close proximity to the 3′ end of the primer;
 13. Akit for use in a method of identifying a base at a target position in anucleic acid which comprises: (a) a polymerase; (b) means for detectingpyrophosphate release; (c) optionally a nucleotide-degrading enzyme; (d)the Sp isomer of an α-thiotriphosphate analogue of an adenine nucleotideand optionally one or more Sp isomers of thymine, cytosine or guaninenucleotides; and (f) optionally, a test specific primer which hybridisesto sample nucleic acid DNA so that the target position is in closeproximity to the 3′ end of the primer; (g) optionally, one or moreunmodified thymine, cytosine or guanine nucleotides; (h) optionally,alkaline phosphatase.